The avian pineal gland is an important model systemfor the study of cellular and molecular bases of circadian clocks and sleep. However, although the mechanisms of the biosynthesis of the major output of this gland, melatonin, are largely understood, the molecular bases for the rhythm generating mechanism has been elusive, and the cellular/molecular basis for its pacemaker properties is completely unknown. Transcriptional profiling and the development of new cell culture methods in the original P01 grant identified several candidate genes that were likely to be important in rhythm generation and showed that melatonin broadly affected metabolic and ionic homeostasis without affecting clock gene rhythms. The present proposal seeks to test the hypothesis that pineal pacemaker activity regulates CNS and peripheral metabolic rhythms via mechanisms that are independent of or at least differentially regulated from output regulation through clock gene rhythms in chicks. (1) We will determine whether surgical disruption of circadian organization by pinealectomy and retinectomy differentially affects glucose metabolism and clock gene rhythms (mRNA and protein) in CNS and peripheral tissues in vivo. We will also ask whether administration of exogenous melatonin differentially affects glucose metabolism and clock genes (mRNA and protein) in CNS and peripheral tissues in vivo by employing both quantitative PCR and DNA microarrays. (2) We have developed a new experimental system in which primary cultured pinealocytes are co-cultured with CNS and peripheral target cells. We will determine the dynamics of pacemaker properties by monitoring melatonin rhythms in the media, and both metabolic activity and clock gene rhythms in pacemaker and target cells simultaneously. Finally, (4) we will determine the roles played by clock genes and other genes identified in our transcriptional profiling by up-regulating and knocking down expression of these and other genes in pacemaker and target cells by lentiviral transduction. Lay Summary: The molecular mechanisms by which the avian pineal generates rhythms of melatonin and by which peripheral tissues respond to melatonin will be studied. The results will provide the first molecular analysis of pineal pacemaker activity. These studies will build a bridge between metabolic and clock gene regulation in sleep-wake cycles in a species whose metabolic demands are more similar to diurnal humans than are those of nocturnal rodents.